20121027 Supplemental Data Final - Plant Cell · Supplemental Data. Ren et al. (2012). Plant Cell...

Supplemental Data. Ren et al. (2012). Plant Cell 10.1105/tpc.111.107144

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SUPPLEMENTAL FIGURES


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Supplemental figure 1. Functional analysis of BP12 lines and sub-cellular

localization of GFP fused FKBP12 protein in Arabidopsis.

(A) The growth of 12 independent BP12 lines and WT control in the presence

and absence of 10 µg/ml rapamycin; 12 independent segregating T2 generation

BP12 lines were initially screened in Basta plates for 5 days; growth of resistant

seedlings at 5 DAG were monitored on 0.5MS+DMSO plate in the absence (left

panel) and presence of 10 µg/ml rapamycin (right panel) for 7 days. Without

rapamycin all BP12 and WT plants display similar root and shoot growth when

grown vertically, whereas with rapamycin in the medium the BP12 lines had

reduced shoot and root growth compared to WT control. (B) 6 DAG WT and four

BP12 lines display no detectable differences when grown on 0.5 MS + DMSO

compared to 0.5 MS medium. (C) DNA-PCR analysis of 17 independent

P35S:FKBP12-GFP transgenic T1 generation plants using 35SF and GFPR

primers show presence of transgene in all lines except line 13; (D) 9 independent

T3 generation lines from Figure A were selected to perform rapamycin sensitive

assay, all show reduced shoot and root growth compared to WT with rapamycin

treatment. WT and P35S:FKBP12-GFP transgenic seedlings at 5 DAG were

treated with rapamycin for 7 days by transferring from 0.5MS medium to 0.5

MS+10µg/ml rapamycin along with control (0.5 MS +1µl/ml DMSO). (E-G)

Confocal images showed the FKBP12-GFP localization and distribution both in

the cytoplasm and nucleus in different tissues from leaf epidermal cells (E),

trichomes (F) and root hairs (G).


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Supplemental figure 2. Molecular identification of transgenics and analysis of

human S6K1 phosphorylation status in Arabidopsis.

(A)The P35S:Hs-S6K construct with kanamycin plant selection marker was

transformed into BP12-2 T3 homozygous plants. DNA-PCR analysis show that

P35S:Hs-S6K1 construct is present in BP12-2 in all 9 independent T1 lines; (B)

Quantitative RT-PCR analysis shows HsS6K gene expression in BP12-2 plants;

(C) ELISA results show that the phosphorylation status of p70 S6K [pT389] was

significantly reduced in response to rapamycin treatment (S6K/BP12-C+RAP),

nutrient starvation (S6K/BP12-C+0.001MS) and dark (S6K/BP12-C+Dark)

compared to normal condition (S6K/BP12-C) : ½ MS media with 22 ºC ,16h/8h

light/dark cycle and 40 µM light intensity. Error bars indicate SD for triplicates; (D)

The ratios of NADH/NAD and NADPH/NADP in BP12 lines do not differ from the

WT in response to rapamycin treatment, indicating that the homeostasis of

NAD(P)H/NAD(P) is able to be maintained in BP12 lines but the relative levels of

NAD(P)H were lower compared to WT (Please see Figure 5E).

troS yawhtaPredrO emaN lacimehcoiByawhtaP buSyawhtaP repuS X

ON 5-21PBON 2-21PBON TW

212 enotecayxordyhid-3,1 74.1131.1222 etahpsohp-6-esotcurf 40.1 22.130.3322 esoculg 29.0 80.1 30.1522 )P6G( etahpsohp-6-esoculg 61.1 3 11.1722 etarecylg 89.0 84.236.2332 etavuryp 88.0 92.1 71.1732 etaratulgotek-ahpla 11.1 20.159.0932 etartic 13.346.219.0042 etaramuf 11.38.28.0242 etalam 80.318.268.0342 )etaoidenetuB-sic( etaelam 78.0 22.185.1642 etaniccus 46.142.178.0

noitaripserotohP462 )etatecayxordyh( etalocylg 21.177.0 83.1082 lotirhtyre 58.0 97.081.1182 *etanorhtyre 78.0 58.149.1382 etanoculg 30.129.058.0882 etahpsohp-5 esolulyx ,etahpsohp-5 esolubir :rabosI 83.158.0 94.0003 lotibir 39.0 86.1 21.1203 esobir 49.030.1 48.0303 esolubir 19.0 72.045.0403 lotierht 8.092.130.1113 etanolyx 89.0 24.126.1213 esolyx 19.0 61.1 10.1

msilobatem lotisonI913 lotisoni-oym 68.0 6.086.0923 etanosolutcoyxoed-3 41.1 2.192.1143 esotcurf 19.0 20.110.1543 esotcalag 88.030.119.0743 etanorutcalag 70.1 81.124.1873 esoniffar 49.0 2.082.0973 esonmahr 19.0 61.1 39.0

msilobatem dica cisabid dehcnarb 5C493 etalamartic 12.1 63.492.4

etardyhobraC

sisylocylG

elcyc ACT

ragus editoelcun dna ragus onimA

msilobatem esotcurf ,esoculg ,esorcuS

.yduts siht ni deliforp slacimehcoib tnacifingis yllacitsitats fo pam taeH deR dna neergp etacidni sllec dedahs ≤ ( 50.0 der ;nosirapmoc taht rof rehgih yltnacifingis era seulav naem eht taht setacidni neerg .)rewol yltnacifingis seulav der thgiL dna

neerg thgil ( 01.0<p<50.0 etacidni sllec dedahs der thgil ;nosirapmoc taht rof rehgih dnert seulav naem eht taht setacidni neerg thgil .)rewol dnert seulav

tnemtaert nicymapaR tuohtiW/htiW

PAR 5-21PBPAR TW PAR 2-21PB

Supplemental Figure 3A. Heat map of biochemicals in the carbohydrate pathway.WT, BP12-2 and BP12-5 seedlings at 15 DAG were treated with rapamycin for 3 days by transferring from 0.5MS medium to 0.5 MS+10 µg/ml rapamycin along with control (0.5 MS +1µl/ml DMSO).Red and green shaded cells indicate p<0.05 (red indicates that the mean values are significantly higher for that comparison; green values significantly lower). Light red and light green shaded cells indicate 0.05<p<0.10 (light red indicates that the mean values trend higher for that comparison; light green valuestrend lower).

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Pathway Sort Order Super Pathway Sub Pathway Biochemical Name X

WT NO BP12-2 NO BP12-5 NO

7 4-guanidinobutanoate 1.33 1.17 1.079 beta-hydroxypyruvate 0.98 1.33 1.213 cysteine 0.91 1.5 1.217 glycine 1.21 1.48 2.2626 N-acetylserine 0.89 1.61 1.9628 O-acetylserine 0.8 1.5 1.6829 serine 1.16 1.44 2.5

31.5 S-methylmethionine 1.22 1.22 1.0638 3-methoxytyrosine 1 1.09 1.1640 4-hydroxycinnamate 1 1.48 1.4165 N-acetyltryptophan 0.73 1.62 1.0773 phenylalanine 1.01 1.53 1.1980 shikimate 0.9 1.02 0.9982 tryptophan 0.99 1.56 1.0983 tyrosine 0.9 1.1 0.986 2-aminoadipate 1.06 1.87 1.5989 alanine 0.73 1.12 1.0791 asparagine 0.98 0.87 1.0692 aspartate 1.05 1.22 1.0993 beta-alanine 0.81 0.78 0.69100 homoserine 1.01 0.99 1.8103 lysine 0.88 1.25 0.91104 methionine 1.04 1.34 1.24

104.5 methionine sulfoxide 1.01 1.39 1.44111 N-acetylmethionine 0.91 1.44 1.28120 pipecolate 1.11 1.85 1.98126 threonine 0.91 1.2 1.31129 2-aminobutyrate 0.83 1.33 1.34130 2-pyrrolidinone 0.87 1.03 1134 4-hydroxybutyrate (GHB) 0.98 2.16 1.62137 arginine 0.96 0.72 0.58141 citrulline 0.91 0.5 0.67142 dimethylarginine (SDMA + ADMA) 0.92 1.62 1.37144 gamma-aminobutyrate (GABA) 0.97 0.89 0.89145 glutamate 1 1.57 1.17147 glutamine 0.97 2.01 2.32149 histidine 0.95 1.98 1.88154 N-acetylglutamate 1.07 0.55 0.76157 N-acetylornithine 1.23 0.67 0.81158 N-acetylproline 1 1.44 0.92159 N-acetylputrescine 1.17 0.99 1.24161 ornithine 1.18 0.6 0.55162 proline 1.05 1.21 1.47163 pyroglutamine* 0.9 1.2 1.52167 trans-4-hydroxyproline 1 1.5 1.19171 Branched Chain Amino Acids (OAA derived) isoleucine 0.99 1.93 1.47174 2-aminopentanoate 0.91 2.07 1.11178 3-hydroxyisobutyrate 0.88 1.05 0.91179 3-methyl-2-oxobutyrate 0.86 0.99 1.11180 3-methyl-2-oxovalerate 0.89 1.34 1.47182 4-methyl-2-oxopentanoate 0.77 1.19 1.51187 leucine 0.96 1.5 1.28196 valine 0.97 1.82 1.61197 5-methylthioadenosine (MTA) 0.88 1.15 1.5198 agmatine 0.88 1.88 2.57201 putrescine 0.97 1.48 1.65202 spermidine 1.02 1.28 1.08204 5-oxoproline 1.2 5.42 4.67207 glutathione, oxidized (GSSG) 0.91 2.09 1.67208 glutathione, reduced (GSH) 1.2 1.48 1.27

Heat map of statistically significant biochemicals profiled in this study.Red and greenshaded cells indicate p≤0.05 (red indicates that the mean values are significantly higher for that comparison; green values significantly lower). Light red and

light green shaded cells indicate 0.05<p<0.10 (light red indicates that the mean values trend higher for that comparison; light green values trend lower).

With/Without Rapamycin treatment

WT RAP BP12-2 RAP BP12-5 RAP

Amino acid

Serine family (phosphoglycerate derived)

Aromatic amino acid metabolism (PEP derived)

Aspartate family (OAA derived)

Glutamate family (alpha-ketoglutarate derived)

Branched Chain Amino Acids (pyruvate derived)

Amines and polyamines

Glutathione metabolism

Supplemental Figure 3B. Heat map of biochemicals in the amino acid pathway and polyamine sub-pathway.WT, BP12-2 and BP12-5 seedlings at 15 DAG were treated with rapamycin for 3 days by transferring from 0.5MS medium to 0.5 MS+10 µg/ml rapamycin along with control (0.5 MS +1µl/ml DMSO).Red and green shaded cells indicate p<0.05 (red indicates that the mean values are significantly higher for that comparison; green values significantly lower). Light red and light green shaded cells indicate 0.05<p<0.10 (light red indicates that the mean values trend higher for that comparison; light green values trend lower).

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831 gamma-glutamylglutamate 0.98 1.02 0.89835 gamma-glutamylisoleucine* 1.16 1.36 1.35836 gamma-glutamylleucine 1.01 1.32 1.37839 gamma-glutamylphenylalanine 1.12 1.72 1.56842 gamma-glutamyltyrosine 0.89 1.8 1.25843 gamma-glutamylvaline 0.98 1.32 1.36

844.1 valylglycine 1.06 0.92 0.77847 alanylisoleucine 0.97 0.93 0.84848 alanylleucine 0.97 0.91 0.92

848.5 alanylphenylalanine 1.03 0.99 0.98849 alanylvaline 0.98 0.94 0.89850 alanyltyrosine 1.06 0.93 0.97853 aspartylphenylalanine 1.03 1.12 0.93860 glycylisoleucine 1.06 1.1 0.73861 glycylleucine 1.03 0.91 0.78

861.1 glycylphenylalanine 0.96 0.92 0.79862 glycylproline 1.09 0.97 0.88

863.1 valylmethionine 1.06 0.93 0.84864 glycyltyrosine 1.09 0.93 0.84865 glycylvaline 0.95 0.94 0.85867 isoleucylisoleucine 1 0.93 0.9868 isoleucylleucine 1.02 0.93 0.92869 leucylisoleucine 1 0.93 0.8

869.2 valylaspartate 1.01 1.18 1.24869.3 valylphenylalanine 0.98 0.99 0.85869.4 valylvaline 0.97 0.93 0.89870 leucylleucine 1.05 0.97 0.88

870.3 isoleucyltryptophan 0.93 1 0.96872.1 leucylglycine 1.08 0.81 0.63

872.13 tyrosylisoleucine 1.03 1.03 0.96872.7 phenylalanylleucine 1.02 1.11 1.02872.8 tyrosylalanine 0.98 0.85 0.76873.1 arginylleucine 0.98 0.91 0.87873.2 isoleucylarginine 1.18 0.94 0.86873.3 isoleucylglutamate 1.04 1.16 1.05873.5 isoleucylglycine 1.09 0.84 0.78873.7 isoleucylserine 1 1.11 0.72873.9 leucylarginine 1.13 1.03 0.99874.1 aspartylleucine 1 1 0.87874.2 histidylleucine 0.95 0.91 0.8874.3 isoleucylalanine 1.04 0.91 0.81874.4 isoleucylvaline 0.99 0.94 0.97874.5 leucylalanine 1.24 0.89 0.86874.6 leucylasparagine 0.97 0.83 0.78874.7 leucylglutamate 1.02 0.88 0.82874.8 leucylphenylalanine 0.93 1.09 0.87874.9 leucylserine 0.99 0.9 0.72875 valylglutamate 0.95 0.92 0.86

875.1 lysylleucine 0.95 0.92 0.84875.2 serylisoleucine* 1 0.91 0.87875.3 serylleucine 0.99 0.91 0.8875.4 serylphenyalanine 0.97 0.91 0.87875.5 threonylleucine 1.01 0.94 0.89875.6 tyrosylleucine 0.94 1.01 0.88875.7 valylisoleucine 0.97 0.94 0.95875.8 valylleucine 0.98 0.97 0.79875.9 phenylalanylserine 0.96 0.97 0.89876 phenylalanylphenylalanine 1.31 0.74 1.19

876.6 arginylisoleucine 1.01 0.81 1.18


light green shaded cells indicate 0.05<p<0.10 ( light red indicates that the mean values trend higher for that comparison; light green values trend lower).



Peptide

gamma-glutamyl

Dipeptide

Supplemental Figure 3C. Heat map of biochemicals in the peptide pathway.WT, BP12-2 and BP12-5 seedlings at 15 DAG were treated with rapamycin for 3 days by transferring from 0.5MS medium to 0.5 MS+10 µg/ml rapamycin along with control (0.5 MS +1µl/ml DMSO).Red and green shaded cells indicate p<0.05 (red indicates that the mean values are significantly higher for that comparison; green values significantly lower). Light red and light green shaded cells indicate 0.05<p<0.10 (light red indicates that the mean values trend higher for that comparison; light green values trend lower).

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Supplemental Figure 3D. Polyamine pathway showing metabolites in red and blue that are significantly up- and down-regulated respectively (p≤0.05). WT, BP12-2 and BP12-5 seedlings at 15 DAG were treated with rapamycin for 3 days by transferring from 0.5MS medium to 0.5 MS+10mg/ml rapamycin along with control (0.5 MS +1ml/ml DMSO).

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921 Amine derived tyramine 1.05 0.83 0.77936 benzoate 0.89 1.23 1.1940 benzoyl-O-glucose 0.92 1.11 0.94952 Fatty acid and sugar derivatives galactarate (mucic acid) 0.95 1.33 1.44970 dihydrokaempferol 0.79 1.11 1.08986 kaempferol 1.03 1.39 1.19987 kaempferol 3-O-beta-glucoside 0.93 0.79 0.92993 naringenin 0.92 0.83 0.71

1000 quercetin-3-galactoside 0.59 0.72 0.961002 quercitrin 1.08 1.24 1.23

1005.4 kaempferol-3-rhamnoside 0.92 1.03 0.991006.1 sulforaphane 1.02 1.14 1.161006.3 3-methylsulfinylpropyl isothiocyanate 1.09 1.15 1.241006.5 sulforaphane-glutathione 1 0.99 1.341006.6 indole-3-carboxylic acid 1.28 1.21 1.181020 5-formylsalicylate 1.06 1.14 1.341023 chlorogenate 1 0.89 1.051025 coniferyl alcohol 1.21 1.51 1.141026 coniferyl aldehyde 0.99 0.78 0.831030 ferulate 1.08 1.35 1.271036 salicylate 1.18 1.18 1.291038 sinapate 1.17 2.64 1.42

1041.1 benzene-1,2,3-triol 0.86 1.75 1.051042 vanillin 1.08 0.95 0.761071 Terpenoids stigmasterol 1.04 0.95 0.91

1073.1 Phytoalexin camalexin 1.3 1.08 1.06


light green shaded cells indicate 0.05<p<0.10 ( light red indicates that the mean values trend higher for that comparison; light green values trend lower).



Secondary metabolism

Benzenoids

Flavonoids

Glucosinolate

Phenylpropanoids

Supplemental Figure 3E. Heat map of biochemicals in the secondary metabolism pathway.WT, BP12-2 and BP12-5 seedlings at 15 DAG were treated with rapamycin for 3 days by transferring from 0.5MS medium to 0.5 MS+10 µg/ml rapamycin along with control (0.5 MS +1µl/ml DMSO).Red and green shaded cells indicate p<0.05 (red indicates that the mean values are significantly higher for that comparison; green values significantly lower). Light red and light green shaded cells indicate 0.05<p<0.10 (light red indicates that the mean values trend higher for that comparison; light green values trend lower).

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403 2-hydroxyglutarate 0.91 3.15 2.63404 2-hydroxymyristate 0.71 0.65 0.81405 2-hydroxypalmitate 0.55 0.87 0.94426 8-hydroxyoctanoate 0.93 1.11 0.77437 azelate (nonanedioate) 1.22 1.27 1.08441 caproate (6:0) 0.85 0.92 0.89442 caprylate (8:0) 1.32 0.85 0.61

511.2 Oxylipins 9,10-hydroxyoctadec-12(Z)-enoic acid 0.72 0.97 0.8523.4 1,2-propanediol 1.01 1.96 0.93544 glycerol 0.95 1.11 0.95558 1-palmitoylglycerophosphoethanolamine 0.82 1.09 0.75559 1-palmitoylglycerophosphoinositol* 0.6 0.64 0.49572 glycerol 3-phosphate (G3P) 1.17 1.55 0.72578 choline 1.07 0.88 0.9579 choline phosphate 1.23 1.22 1.06580 ethanolamine 0.98 0.97 0.97598 beta-sitosterol 0.92 1.05 0.79601 campesterol 0.96 1.06 0.81622 Cutin and cuticular 3-hydroxybutyrate (BHBA) 0.77 1.62 1.57643 CoA metabolism pantothenate 1.02 1.24 1.09653 nicotinamide 1.36 1.17 1.19654 nicotinamide adenine dinucleotide (NAD+) 0.93 1.28 1.06658 nicotinamide ribonucleotide (NMN) 1.23 1.74 1.02659 nicotinamide riboside* 1.38 1.49 1.3660 nicotinate 1.06 1.01 0.8663 nicotinate ribonucleoside* 0.97 0.95 0.88667 trigonelline (N'-methylnicotinate) 0.75 0.84 0.85677 methylphosphate 0.9 0.99 0.73678 phosphate 1.03 1.51 1.06685 Riboflavin and FAD metabolism riboflavin (Vitamin B2) 0.93 0.92 0.88689 Quinone metabolism phytonadione (Vitamin K1) 1.08 0.98 0.8692 ascorbate (Vitamin C) 0.98 1.15 0.99693 dehydroascorbate 0.93 1.02 0.94705 Thiamine metabolism thiamin (Vitamin B1) 0.93 0.81 1.04707 Tocopherol metabolism alpha-tocopherol 1.15 1.84 0.94716 pyridoxal 1.03 0.96 0.82720 pyridoxate 1.05 1.11 0.99727 pheophorbide A 0.69 0.82 1.79

729.5 phytol 0.96 1.16 0.83737 2'-deoxyadenosine 0.91 0.93 0.95747 adenine 1.08 0.93 0.86748 adenosine 1.11 0.95 0.92749 adenosine 2'-monophosphate (2'-AMP) 1 1.13 0.86752 adenosine 3'-monophosphate (3'-AMP) 0.97 0.89 1756 adenosine-2',3'-cyclic monophosphate 0.91 1.15 0.83

756.5 N6-carbamoylthreonyladenosine 0.93 1.17 1.09759 allantoin 1.03 2.2 1.13762 guanine 1.03 0.75 0.97763 guanosine 0.95 0.6 0.72768 guanosine-2',3'-cyclic monophosphate 0.97 1 0.91773 N1-methyladenosine 0.94 0.89 0.99774 N1-methylguanosine 0.72 1.06 0.99780 urate 0.96 1.83 1.21

784.1 guanosine 3'-monophosphate (3'-GMP) 0.89 1.58 1.04786 2'-deoxycytidine 1.27 1.53 1.25793 3-aminoisobutyrate 1.05 1.47 1.09800 5-methylcytidine 0.99 1.33 2.22

801.4 5-methyluridine (ribothymidine) 0.89 0.88 0.97802 cytidine 0.99 0.73 0.89806 cytidine-3'-monophosphate (3'-CMP) 1.03 1.07 0.99808 cytosine-2',3'-cyclic monophosphate 0.93 1.2 1.02812 orotate 0.89 1.61 1.01815 pseudouridine 0.93 0.72 0.87816 thymidine 1.16 0.91 0.84823 uridine 0.95 0.85 0.9

Nucleotide

Purine metabolism

Pyrimidine metabolism

Lipids

Free fatty acid

Cofactors, Prosthetic Groups, Ele

Nicotinate and nicotinamide metabolism

Oxidative phosphorylation

Ascorbate metabolism

Vitamin B metabolism (B6 or B12)

Chlorophyll and heme metabolism

Glycerolipids

Phospholipids

Choline metabolism

Sterols

Heat map of statistically significant biochemicals profiled in this study. Red and green shadedcells indicate p≤0.05 (red indicates that the mean values are significantly higher for that comparison; green values significantly lower). Light red and light green

shaded cells indicate 0.05<p<0.10 (light red indicates that the mean values trend higher for that comparison; light green values trend lower).


BP12-5 RAPWT RAP BP12-2 RAP

Supplemental Figure 3F. Heat map of biochemicals in the lipid, cofactors and nucleotide pathway.WT, BP12-2 and BP12-5 seedlings at 15 DAG were treated with rapamycin for 3 days by transferring from0.5MS medium to 0.5 MS+10 µg/ml rapamycin along with control (0.5 MS +1µl/ml DMSO).Red and green shaded cells indicate p<0.05 (red indicates that the mean values are significantly higher for thatcomparison; green values significantly lower). Light red and light green shaded cells indicate 0.05<p<0.10(light red indicates that the mean values trend higher for that comparison; light green values trend lower).

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Supplemental Figure 4. Gene expression levels in rapamycin treated BP12-2

plants compared to WT.

The graph shows comparative levels of transcripts of selected genes in the RNA-

Seq data and validation by real time-PCR. WT, BP12-2 and BP12-5 seedlings at

15 DAG were treated with rapamycin for 3 days by transferring from 0.5MS

medium to 0.5 MS+10 g/ml rapamycin along with control (0.5 MS +1µl/ml DMSO).

EB3-EXPANSIN B3; EA8-EXPANSIN A8; ET6-Extensin 6; ET11-Extensin 11;

PL-pectate lyase; FLA6-FASCICLIN-LIKE ARABINOGALACTAN 6; GLP-

glycoprotein family protein; SPL5-SPIRAL1-LIKE5; RHS15-ROOT HAIR

SPECIFIC 15; MRH6-MORPHOGENESIS OF ROOT HAIR 6; PT2-PHOSPHATE

TRANSLOCATOR 2; IRT1-IRON-REGULATED TRANSPORTER 1; NDK2-

NUCLEOSIDE DIPHOSPHATE KINASE 2; RBCS1B-RuBisCO small subunit 1B

(RBCS-1B); RBCS3B-RuBisCO small subunit 3B (RBCS-3B); GS3-galactinol

synthase 3; G6P-GLUCOSE-6-PHOSPHATE/PHOSPHATE TRANSLOCATOR 2;

SPS7-SUCROSE-PROTON SYMPORTER 7; LHT7-LYS/HIS TRANSPORTER 7;

LHT1-LYSINE HISTIDINE TRANSPORTER 1; ERD5-EARLY RESPONSIVE TO


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DEHYDRATION 5; SAD-S-adenosylmethionine decarboxylase; AOX1D-

alternative oxidase 1D (AOX1D);GDH-GLUTAMATE DEHYDROGENASE. Error

bars indicate SD for triplicates.


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Supplemental figure 5. Rapamycin treatment at pre-flowering stage delayed the

growth rate and phase change in BP12-2 plants.

(A) Phenotypic analysis of BP12-2 and WT grown on MS media without

rapamycin for 35 DAG show similar growth in both lines; (B) BP12-2 and WT

plants were grown on MS media for 25 DAG and then transferred to

MS+rapamycin media; after 10 DAG of growth the WT plants show

bolting/flowering whereas the BP12-2 plants remain in the vegetative phase.


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Supplemental figure 6. Molecular identification of rps6a and rps6b mutants.

(A) Genomic structure of the RPS6A and RPS6B genes of Arabidopsis and their

corresponding T-DNA insertion sites. Shaded boxes indicate exons; open boxes

indicate UTR and orange arrows indicate primer position and orientation. (B) Molecular identification of rps6a and rps6b T-DNA insertion mutants by DNA

PCR (1 and 3 respectively) and RT-PCR (2 and 4 respectively). Actin2 was used

as control in the RT-PCR experiments.


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Supplemental Figure 7. rps6 mutants show nutrition and light dependent growth

performance. (A) Comparison of 12 DAG rps6a and rps6b mutant and WT seedlings grown

under different light (10 µM and 100 µM) and nutrition (0.001x, 0.5x and 2.0xMS)

conditions. Under nutrition starvation conditions (0.001xMS) with 10 µM or 100

µM light intensity, the seedling growth of rps6a and rps6b is slightly better than

WT (upper panel); with 0.5xMS, the seedling growth is relatively better in both

rps6a and rps6b compared to WT under 10 µM light intensity, whereas under 100

µM light intensity the mutants seedling growth is significantly slower (middle

panel); with 2.0xMS, the rps6a and rps6b seedling growth is much slower than

WT under both 10 µM and 100 µM light conditions; (B and C) With increasing of

light intensity and nutrition concentration, the fresh weight of rps6a and rps6b

accordingly decreased when compared to WT; (B) Under strong light condition

(100 µM), the fresh weight of rps6a and rps6b are lower than that of WT in rich

nutrition condition (0.5, 1.0 and 2.0xMS) and higher than that of WT in poor

nutrition condition (0.001, 0.01 and 0.1xMS); (C) Under weak light condition,

except grown on 1xMS and 2xMS media, the fresh weight of rps6a and rps6b are

higher than that of WT grown on 0.001xMS to 0.5xMS. Error bars indicate SD for

triplicates.


Supplemental Figure 8. Molecular identification of RPS6A-OE1 and RPS6B-OE1

transgenic plants by DNA PCR, RT-PCR and immunoblot analyses.

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Supplemental Figure 9. TOR, RPS6A and RPS6B overexpression result in early

onset of senescence.

(A-C) Phenotypic analysis of TOR, RPS6A and RPS6B overexpression in the soil

under 200 µM light intensity (upper panel) and in vitro under 100 µM light

intensity on MS plates (bottom panel) showed relatively faster growth and early

senescence in the respective overexpression lines compared to WT. Analyses of

these lines at different time points (16-40 DAG) for chlorophyll content (D), cell

death rate (E), SAG12 expression (F) and SAG13 expression (G) also showed

earlier loss of chlorophyll, higher cell death rate, and higher expression of

SAG12/SAG13 markers in TOR-OE1 followed by the RPS6A-OE1 and RPS6B-

OE1 compared to WT. The SAG12/SAG13 expression levels in WT are

presented as 1. Error bars indicate SD for triplicates.


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Supplemental Figure 10. Levels of TOR and RPS6 determine the rate of protein

synthesis as measured by GUS reporter expression in Arabidopsis.

The P35S:GUS construct was introduced into WT, TOR-OE1,RPS6A-OE1,

RPS6B-OE1, rps6a, rps6b and BP12-2 backgrounds. The rate of protein

synthesis was estimated using GUS as a reference. In BP12-2 line, 12 hr

rapamycin treatment was given before measuring GUS levels. The protein

synthesis rate estimated in this study showed the highest level in TOR-OE1

followed by RPS6A-OE1 and RPS6B-OE1, whereas the lowest protein synthesis

rate was observed in the rapamycin treated BP12-2 followed by rps6b and rps6a

mutant lines. Error bars indicate SD for triplicates.


21

Supplemental Figure 11. Proposed mechanism of rapamycin-induced redox and metabolite alterations in BP-12 plants. Solid lines indicate direct experimental evidence from our work and dashed lines indicate evidence as suggested by the literature. Based on our analysis of changes in redox status and metabolite profile, we propose that TOR activity inhibition in BP12 plants by rapamycin leads to an immediate increase in oxygen consumption and thereby oxygen depletion, similar to what is reported in yeast (Bonawitz et al., 2007) and mammalian cells (Ramanathan and Schreiber, 2009). Reactive oxygen species (ROS) such as H2O2 are inevitable byproducts of aerobic energy metabolism via the cytochrome pathway and are enhanced under hypoxia (Baxter-Burril et al., 2002; Subbaiah, 2009). In plant cells, multiple mechanisms come into play to alleviate ROS-mediated damage and prolong cell survival. Transcriptome and metabolite profiling data indicates that there are 3 major pathways that are activated under rapamycin treatment, that lead to reduction in ROS levels in rapamycin treated plants (Figure 5D; root hair assay). 1) The induction of alternative oxidase by rapamycin (AOX1D induction by >2-fold, Supplemental Figure 4 online: QRT-PCR data), which directly transfers electrons from the ubiquinone to oxygen, prevents over-reduction of the cytochrome pathway and thereby the production of ROS. Enhanced uncoupled respiration by the induction of AOX activity also allows the maintenance of TCA cycle and production of carbon skeletons needed for amino acid and nucleotide biosynthesis. 2) A 3-fold increase in total glutathione content and the maintenance of ascorbate levels (as shown by our metabolite analysis, Figure 3;


22

Supplemental Figure 3F online) can reduce and detoxify H2O2. 3) Hypoxia also leads to nitric oxide (NO) generation via nitrite reduction in plants (Gupta and Igamberdiev, 2011) and NO is a scavenger of ROS including the highly damaging superoxide species (de Oliveira et al., 2008; Tossi et al., 2011). The potential induction of NO by rapamycin is suggested by the metabolite profile. Aconitase inhibition by NO is known to enhance the level of organic acids such as citric acid (which in turn activates AOX) and amino acids (Gupta et al., 2011) and AOX1D induction under AOX1a-downregulated was also shown to shift the metabolism towards amino acid biosynthesis in Arabidopsis (Strodkotter et al., 2009). Both NO and AOX induction also promote phenylpropanoid biosynthesis (Tossi et al., 2011; Sircar et al., 2012). The enhanced uncoupling capacity provided by the alternative oxidase and the consequent alleviation of ROS in yeast and plants seems to be important for the enhanced lifespan in response to TOR inhibition in these two phyla (our results; Pan and Shadel, 2009). Most mammals lack AOX and their response to enhanced oxygen consumption induced by the suppression of TOR activity is contrasting (Ramanathan and Schreiber, 2009). Inhibiting mTOR function rapidly decreases uncoupled respiration and induces organic acid and amino acid starvation, culminating in decreased cell viability. References: Baxter-Burrell, A., Yang, Z., Springer, P.S., Bailey-Serres, J. (2002) RopGAP4-dependent Rop-GTPase rheostat control of Arabidopsis oxygen deprivation tolerance. Science 296: 2026-2028. Gupta KJ, Igamberdiev AU. 2011. The anoxic plant mitochondrion as a nitrite: NO reductase. Mitochondrion 11: 537–543. Gupta KJ, Shah JK, Brotman Y, Jahnke K, Willmitzer L, Kaiser WM, Bauwe H, Igamberdiev AU. 2012. Inhibition of aconitase by nitric oxide leads to induction of alternative oxidase and to a shift of metabolism towards biosynthesis of amino acids. Journal of Experimental Botany 63: 1773–1784. Pan Y., Shadel G. S.2009 Extension of chronological life span by reduced TOR signaling requires down-regulation of Sch9p and involves increased mitochondrial OXPHOS complex density. Aging 1, 131–145. Ramanathan A, Schreiber SL (2009) Direct control of mitochondrial function by mTOR. Proc Natl Acad Sci USA 106: 22229–22232. Sircar D, Cardoso HG, Mukherjee C, Mitra A, Arnholdt-Schmitt B. (2012) Alternative oxidase (AOX) and phenolic metabolism in methyl jasmonate-treated hairy root cultures of Daucus carota L. J Plant Physiol. 169:657-63. Strodtkötter I, Padmasree K, Dinakar C, Speth B, Niazi PS, Wojtera J, Voss I, Do PT, Nunes-Nesi A, Fernie AR, Linke V, Raghavendra AS, Scheibe R. (2009) Induction of the AOX1D isoform of alternative oxidase in A. thaliana T-DNA insertion lines lacking isoform AOX1A is insufficient to optimize photosynthesis when treated with antimycin A. Mol Plant. 2:284-97 Subbaiah CC (2009) Ionic loops and rebounds: oxygen-deprivation signaling in plants. In: Mancuso S, Baluśka F (eds) Signaling in plants. Springer, Berlin, pp 195–207. Tossi V, Amenta M, Lamattina L, Cassia R. (2011) Nitric oxide enhances plant ultraviolet-B protection up-regulating gene expression of the phenylpropanoid biosynthetic pathway. Plant Cell Environ. 34:909-21.


23

Supplemental Tables Supplemental Table 1. Different concentrations of MS medium used in this study.

MS medium types Sucrose (G/L)

MS macro- micronutrients (G/L) PH

2xMS 60 G 8.8G 5.8 1XMS 30G 4.4G 5.8 0.5XMS 15G 2.2G 5.8 0.1XMS 3G 0.44G 5.8 0.01XMS 0.3G 0.044G 5.8 0.001XMS 0.03G 0.0044G 5.8

Note: Sucrose obtained from Anachemia, Cat No is 87688-380. MS macro- &

micronutrients purchased from Caisson, Cat No is MSPC0630 Supplemental Table 2. Different nitrogen concentration used in MS medium.

Medium types Sucrose (G/L) NH4NO3 (G/L) KCl (G/L) PH MS-N 30G 0G 1.9G 5.8 MS+0.01GN 30G 0.01G 1.9G 5.8 MS+0.1GN 30G 0.1G 1.9G 5.8 MS+0.5GN 30G 0.5G 1.9G 5.8 MS+1GN 30G 1G 1.9G 5.8 MS+2.5GN 30G 2.5G 1.9G 5.8 MS+5GN 30G 5G 1.9G 5.8 MS+7.5GN 30G 7.5G 1.9G 5.8


24

Note: KNO3 was replaced by KCl in the medium, and GN is grams nitrogen

added to the medium. The other composition is same as the MS (Murashige and

Skoog, 1962).


25

Supplemental Table 3. Different sucrose concentration used in the MS medium.

Medium types Sucrose (G/L) MS macro- Micronutrients (G/L) PH

MS-C 0G 4.4G 5.8

MS+0.1GC 0.1G 4.4G 5.8

MS+0.5GC 0.5G 4.4G 5.8

MS+5GC 5G 4.4G 5.8

MS+10GC 10G 4.4G 5.8

MS+15GC 15G 4.4G 5.8

MS+30GC 30G 4.4G 5.8

Note: GC stands for grams sucrose added to the medium. Sucrose purchased from Anachemia, Cat No is 87688-380. MS macro- &

micronutrients ordered from Caisson, Cat No is MSPC0630.


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Supplemental Table 4. Analysis of GFP expression levels of PrRNA:GFP in WT and BP12-2 background under different nutrition and

rapamycin treatment conditions.

Constructs MS MS+

Rap MS+Dark 0.001MS MS-C MS-N MS+Dark+Rap 0.001MS

+Rap 0.001MS +Rap +Dark

PrRNA:GFP/WT 1 ± 0.09 1 ± 0.08 0.26 ± 0.03 0.17 ± 0.01 0.27 ± 0.02 0.19 ± 0.01 0.27 ± 0.02 0.17 ± 0.01 0.14 ± 0.01

PrRNA:GFP/BP12-2

1 ± 0.1 0.19 ± 0.02 0.26 ± 0.02 0.18 ± 0.01 0.26 ± 0.02 0.20 ± 0.01 0.18 ± 0.01 0.13 ± 0.01 0.13 ± 0.01

Note: The values showing relative expression of GFP were quantified by real time PCR. The GFP transcription level in

PrRNA:GFP/WT Arabidopsis grown on MS was set as control (value=1) and the relative GFP expression of PrRNA:GFP/WT and

PrRNA:GFP/BP12-2 Arabidopsis grown on various conditions are shown in the table as fold change compared to the control. Errors

indicated ±SD for triplicates


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Supplemental Table 5. Growth stages and leaf numbers of BP12-2, rps6a, rps6b, rps6b/BP12-2, TOR-OX1 and TOR-OX1/BP12-2

grown on MS+rapamycin plates compared to WT control.

Arabidopsis lines Hypocotyl and cotyledon emergence (DAG)

Second rosette leaf appearance (DAG)

First flower buds visible (DAG)

First flower open (DAG)

Plants death (DAG)

Number of rosette leaves (count)

WT 2.1 ± 0.2 7.5 ± 0.8 23.4 ± 2.7 28 ± 2.1 66.4 ± 6.5 11.2 ± 2.7

BP12-2 3.4 ± 0.4 15.6 ± 1.1 36.8 ± 4.1 43 ± 3.3 83.8 ± 7.5 13.4 ± 3.3

Rps6a 2.8 ± 0.3 9.2 ± 0.7 28.3 ± 3.4 34 ± 2.6 72.3 ± 6.3 11.7 ± 2.7

Rps6b 2.9 ± 0.2 9.8 ± 1.1 29.1 ± 3.7 35 ± 3.1 74.1 ± 8.2 12.2 ± 3.1

Rps6b/BP12-2 3.3 ± 0.4 11.3 ± 1.3 34.4 ± 2.8 39 ± 3.4 78.4 ± 6.1 12.8 ± 3.4

TOR-OE1 2.0 ± 0.3 6.7 ± 0.8 20.2 ± 2.3 24.1 ± 3.2 59.3 ± 6.7 9.4 ± 3.1

TOR-OE1/BP12-2 2.1 ± 0.3 7.2 ± 0.7 22.8 ± 2.5 27.2 ± 2.3 65.6 ± 7.3 10.8 ± 2.3

Note: Values shown are mean number ± SD (standard deviation) from 20 independent Arabidopsis plants. Growth condition: 16h/8h

light/dark cycle, 22 ºC, light intensity=100 µM. DAG=days after germination.


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Supplemental Table 6. Growth stages and leaf numbers of rps6a, rps6b, TOR-OE1, RPS6A-OE1 and RPS6B-OE1 and WT lines grown

in soil.

Arabidopsis lines

Hypocotyl and cotyledon emergence(DAG)

Second rosette leaves appearance(DAG)

First flower buds visible (DAG)

First flower open (DAG)

First silique ripening (DAG)

Seeds ready to harvest (DAG)

Numbers of rosette leaves (count)

Wild Type 2.5 ± 0.4 8.1 ± 1.3 23.2 ± 2.7 28.2 ± 2.8 45.3 ± 3.4 71.4 ± 4.5 14.2 ± 1.2

Rps6a 2.8 ± 0.1 9.2 ± 1.4 26.5 ± 2.5 32.7 ± 3.6 50.6 ± 4.7 76.7 ± 5.3 15.1 ± 1.6

Rps6b 2.9 ± 0.2 9.5 ± 1.6 27.9 ± 2.3 33.6 ± 4.2 51.2 ± 4.9 78.3 ± 5.1 15.4 ± 1.4

TOR-OX1 2.1 ± 0.1 7.2 ± 1.5 19.3 ± 2.0 23.4 ± 3.4 40.8 ± 3.8 64.2 ± 3.8 11.4 ± 2.2

RPS6A-OX1 2.2 ± 0.2 7.5 ± 1.4 20.4± 2.2 25.6 ± 4.1 42.9 ± 4.6 66.5 ± 4.5 12.1 ± 1.8

RPS6B-OX1 2.3 ± 0.4 7.8 ± 1.2 20.8 ± 1.9 26.7 ± 4.4 43.7 ± 5.3 67.1 ± 3.9 12.6 ± 1.9

TOR-

OX1/rps6b

2.4 ± 0.3 7.8 ± 1.1 22 ± 2.3 26.4 ± 2.1 43 ± 4.4 68.3 ± 5.1 13.5 ± 1.3

Note: Values shown are mean number ± SD (standard deviation) from 20 independent Arabidopsis plants. Growth condition: 16h/8h

light/dark cycle, 22 ºC, light intensity= 200 µM. DAG=days after germination.


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Supplemental Table 7. Expression levels of transgenes in TOR-OE1, RPS6A-OE1 and RPS6B-OE1 Arabidopsis lines.

CONSTRUCTS Transgene expression levels (compared to WT): Fold

change

TOR-OE1 58.6 ± 8.3

RPS6A-OE1 37.2 ± 7.1

RPS6B-OE1 32.7 ± 5.7

Note: The values represent relative expression levels in respective transgenic lines compared to WT control. The total RNA was

isolated from leaves of 10 DAG plants. The real time PCR primers used in the expression analysis are listed in Supplemental Table S11.

Actin2 was used as internal control. Errors indicate ±SD for triplicates.


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Supplemental Table 8. Characterization of T-DNA insertion lines and their phenotypes.

Alleles Gene number SALK lines

Insertion sites

Truncated CDS

rps6a At5g10360 SALK_012147 fourth exon ATG+18 bp

rps6b AT4G31700.1 SALK_061539

second

exon ATG+120

Note: The salk lines were ordered from ABRC and the knockout lines were identified by PCR with primers designed from T-DNA Primer

Design website: http://signal.salk.edu/tdnaprimers.2.html

Supplemental Table 9. PCR primers used for identification of SALK knockout

lines.

Primer name Primer sequence

Rps6aL CTGCATCAGAGTTAGCCTTGG

Rps6aR TGAATCCAAACCTTTGAATCG

Rps6bL GTATTCATTCACCATCACCCG

Rps6bR AAATGGAATCTGTACCTCGCC

Supplemental Table 10. PCR primers used for cloning.

Primers name Primers sequence

RPS6AF GCGGCCGCATGAAGTTCAACGTCGCCAATCC

RPS6AR CCCGGGTTAAGCAGCAACGGGTTTAGCAGGAG

RPS6BF GCGGCCGCATGAAGTTCAACGTTGCGAATC

RPS6BR CCCGGGTTAAGCTGTGACAGAGGGCTTGGCA

PRPS6BF GCGATCGCATGTGATTGTGCGGTTGTTGATG

PRPS6BR GCGGCCGCCTTCGACGTCTAGGGTTTCTCTG

PRPS6AF GCGATCGCTTGAGCGGCGGAGAAGATTGTG

PRPS6AR GCGGCCGCCGTCTCCGATAGACACTGCTGC

ScFKBP12F GCGGCCGCATGACCGAACATGAGTCTTTAG

ScFKBP12R CCCGGGTTAGTTGACCTTCAACAATTCG

GUSF GCGGCCGCATGTTACGTCCTGTAGAAACC

GUSR CCCGGGTCATTGTTTGCCTCCCTGCTGC

PTORF GCGATCGCGTTTAAACCGAAAGAGCAAG

PTORR GCGGCCGCCTAGCACTTCCATATAGATAAT


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Supplemental Table 11. Primers used for Real time PCR

Gene (accession #) Primer name Primer sequence

SAG12 (AT5G45890) RLSAG12F TGATGAGCAAGCACTGATGARLSAG12R TGCACTCTCCAGTGAACACA

RPS6A (AT4G31700) RLRPS6AF CGACACTGAGAAGCCAAGAARLRPS6AR GAAGGTACGGCGGTAAGTGT

yeast FKBP12 (M57967) RLScFKBP12F GTCATCAAGGGTTGGGATGTRLScFKBP12R AATCAAACCTGGGAAACCAC

RPS6B (AT5G10360) RLRPS6BF ACCAAAGAGAGCCTCCAAGARLRPS6BR TTGCTAACTTCCTTGCCCTT

ACTIN2 (AT3G18780) RLACTIN2F GCTGACCGTATGAGCAAAGARLACTIN2R ATCTGCTGGAATGTGCTGAG

SAMDC (AT3G02470) RLSADF TGGGCAATGATGATGAGACTRLSADR GTTGTTGCAGTTGCTGGAGT

RHS15 (AT4G25220) RLRHS15F TCGACTTGGCTTTCCTCTCTRLRHS15R CATACCGATGGTCAGAAACG

RBCS3 (AT5G38410) RLRBCS3BF GACTCCGCTCAAGTGTTGAARLRBCS3BR TGTCGAATCCGATGATCCTA

RBCS1 (AT1G67090) RLRBCS1BF GACTCCGCTCAAGTGTTGAARLRBCS1BR TGTCGAATCCGATGATCCTA

PT2 (AT2G38940) RLPT2F TTATCCTTGGAGACCGAACCRLPT2R TCCAAATCTCCTCCTTCCAC

RHS14 (AT4G22080) RLPLF TGTGGAGAGAATGCCAAGAGRLPLR CGCTACCACCAATTGCATAC

NDPK2 (AT5G63310) RLNDPK2F AGAATTGGCTGAGGAGCATTRLNDPK2R GAAGCAACAACACCAACACC

MRH6 (AT2G03720) RLMRH6F GGAGGAGGAGTGGTCGAGTARLMRH6R GACGTTTCGTGGTGATCAAG

LHT1 (AT5G40780) RLLHT1F TGATCTTTGCTTCGGTTCACRLLHT1R TCTTGAACACCTTTGCTTGC

LHT7 (AT4G35180) RLLHT7F TCCTTCCGATTCAAAGAACCRLLHT7R CCGGAATCTTGTCTCCGTAT

SUC2 (AT1G22710) RLSPS7F TATCTTCGCTCTCGGGTTCTRLSPS7R CTCCAGCTGCTAAATCACCA

SP1L5 (AT4G23496) RLSPL5F CAAAGAGTTCAGGGCCAAARLSPL5R CAGGAACCGATTTCACCTTC

IRT1 (AT4G19690) RLIRT1F ATTTACATGGCACTCGTGGARLIRT1R AGAGCCGCGATTAAACACTT

GLP (AT5G26080) RLGPPF CTCTAGGAGTTTTCTACACCCRLGPPR CCAAGGTTGTCCGGAGGGGG

GDH2 (AT5G07440) RLGDHF GACGCAACTGGAAGTCTCAARLGDHR TTTCCTTGTTCAGGACACCA

GolS3 (AT1G09350) RLGS3F ATGGCACCTGAGATGAACAARLGS3R TTTCCTTAGCCCTTTAGCCA

GPT2 (AT1G61800) RLG6PT2F GTGGGTAGTGGCACAAAGTGRLG6PT2R ATCGATGCAACAATAACGGA

FLA6 (AT2G20520) RLFA6F ACAAGCACAAAGCAACCAAGRLFA6R CAACAACACACTGTCCACCA

EXT11 (AT5G49080) RLEXT11F TCCCTCACCAAAGGTTGACTRLEXT11R AGTACGGTGGAGGTGGAGAG

EXT6 (AT2G24980) RLEXT6F TACTCGCCTTCTCCAAAGGTRLEXT6R CGAGTAGTACGGTGGTGGTG

EXPB3 (AT4G28250) RLEXPB3F ATGGAGAAGGAGACATTGGCRLEXPB3R ATGGAGAATGGTCCCTTGAG

EXPA8 (AT2G40610) RLEXPA8F CGCGTGCTATGAGATGAAGTRLEXPA8R AAGAGGAGGATTGCACCAAC

ERD5 (AT3G30775) RLERD5F TTCTCCGAATCGAGTCCTCTRLERD5R ATTGGACTCTTGGCATTTCC

AOX1D (AT1G32350) RLAOX1DF CATCTCCGGTAAGTGGGAATRLAOX1DR CCGTCCGGTTTAGTGATCTT


33

Supplemental Methods:

Identification of RPS6 single and double mutants in Arabidopsis:

The RPS6 is represented by two isoforms, RPS6A (At5g10360) and RPS6B

(AT4G31700) and these are mapped on chromosome 4 and 5 respectively

(Supplemental figure 6A) in Arabidopsis genome. The rps6a allele contained a

T-DNA insertion in the fourth exon, 189 bp downstream ATG of CDS

(Supplemental figure 6A). The rps6b insertion alleles disrupted by T-DNA in the

second exon downstream of ATG (Supplemental figure 6A).These two RPS6

isoforms share 85% and 95% identity in DNA and protein sequence level,

respectively. T-DNA insertional lines of rps6a and rps6b have been confirmed by

PCR (Supplemental figure 6B online). In the homozygous lines, the expression

of the corresponding transcripts was not detected by RT-PCR (Supplemental

figure 6B online). Compared to WT, the rps6a, rps6b insertion alleles showed

slower growth rate, long lived, and also displayed additional morphological

phenotypes (Figure 8A and 8B): vegetative rosette leaves of rps6 are narrow,

pointed and spatulate shape; changes in vascular pattern; slow root and shoot

growth; reduced leaf size and overall plant body size; late flowering; delayed

transition from vegetative growth to reproductive growth (Figure 8A and 8B).

Additionally, also observed leaf vascular and root pattern defects in rps6 mutant

(Figure 8D and 8E). Similar observation have been made in a recent study (Creff

et al., 2010), however in the present study we observed stronger phenotypes

with rps6b.

To gain further insights into RPS6 functions in plants, we constructed

rps6a/rps6b double mutant through reciprocal cross between rps6a and rps6b

homozygous mutants by PCR-based genotyping. All of 54 F1 generation plants

that contain +/rps6a+ /rps6b genotype exhibited same phenotype as rps6a or

rps6b single mutant (Figure 8C). These results imply that rps6 isoforms exhibited

a clear gene dosage effect and at both copies of RPS6 were required for normal

plant development in Arabidopsis. In F2 and F3 generations, we tried to identify

the double mutant through both genotyping by PCR analysis and phenotyping by


34

screening the rps6a and rps6b-like or more severe phenotypes, but we failed to

obtain the double mutants likely due to strong lethality as also observed in the

recent report (Creff et al., 2010).

Complementation tests of RPS6A and RPS6B:

The identity of RPS6A and RPS6B was confirmed by complementation test.

2,632bp genome fragment containing the RPS6A coding sequence and 1.2kb

promoter sequence was transformed into rps6a mutant plants. T1 seeds were

screened on basta MS plates for 7 days. 81 T1 basta-resistant plants were

transferred to soil. All of these T1 plants showed full complementation of rps6

mutant phenotypes (Figure 8F1). 20 of these lines were selected to identify both

homozygous rps6a knockout site and basta marker gene through performing

PCR. In the T2 segregation population, majority showed segregation ratio of 1

mutant: 3 wild-type and the basta gene co-segregated with WT plants, which

confirmed the identity of rps6a mutant gene. In parallel, the same approach was

employed for rps6b complementation test, phenotyping and genotyping of this

mutant allele. As predicted, 3,563bp genome fragment of RPS6B coding and

promoter sequence complemented rps6b mutant (Figure 8F2). Since the

knockout of RPS6A or RPS6B showed similar phenotypes, we examined

whether they have redundant or dose-dependent functions. We therefore

performed the reciprocal complementation test by using 2,632bp genome

fragment of RPS6A to transform rps6b knockout plants and 3,563bp genome

fragment of RPS6B to transform rps6a mutant plants. The results suggested that

RPS6A can rescue rps6b knockout plants and RPS6B is able to complement

rps6a mutant phenotypes (Figure 8F3-4), suggesting RPS6A and RPS6B are

paralogues with similar functions in Arabidopsis.

Creff, A., Sormani, R., and Desnos, T. (2010). The two Arabidopsis RPS6

genes, encoding for cytoplasmic ribosomal proteins S6, are functionally

equivalent. Plant Mol Biol 73, 533-546.

Kim, S.-Y., and Volsky, D. (2005). PAGE: Parametric Analysis of Gene Set

Enrichment. BMC Bioinformatics 6, 144.

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